Skeletal muscle proteome expression differentiates severity of cancer cachexia in mice and identifies loss of fragile X mental retardation syndrome-related protein 1.

Tandem mass tag (TMT)-based quantitative proteomics was used to examine protein expression in skeletal muscle from mice with moderate and severe cancer cachexia to study mechanisms underlying varied cachexia severity. Weight loss of 10% (moderate) and 20% (severe) was induced by injection of colon-26 cancer cells in 10-week old Balb/c mice. In moderate cachexia, enriched pathways reflected fibrin formation, integrin/mitogen-activated protein kinase (MAPK) signaling, and innate immune system, suggesting an acute phase response and fibrosis. These pathways remained enriched in severe cachexia; however, energy-yielding pathways housed in mitochondria were prominent additions to the severe state. These enrichments suggest distinct muscle proteome expression patterns that differentiate cachexia severity. When analyzed with two other mouse models, eight differentially expressed targets were shared including serine protease inhibitor A3N (Serpina3n), synaptophysin-like protein 2 (Sypl2), Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial (Idh3a), peroxisomal acyl-coenzyme A oxidase 1 (Acox1), collagen alpha-1(VI) chain (Col6a1), myozenin 3 (Myoz3), UDP-glucose pyrophosphorylase (Ugp2), and solute carrier family 41 member 3 (Slc41a3). Acox1 and Idh3a control lipid oxidation and NADH generation in the TCA cycle, respectively, and Col6a1 comprises part of type VI collagen with reported profibrotic functions, suggesting influential roles in cachexia. A potential target was identified in fragile X mental retardation syndrome-related protein 1 (FXR1), an RNA-binding protein not previously implicated in cancer cachexia. FXR1 decreased in cachexia and related linearly with weight change and myofiber size. These findings suggest distinct mechanisms associated with cachexia severity and potential biomarkers and therapeutic targets.

Mitochondrial dynamics and quality control are altered in a hepatic cell culture model of cancer cachexia.

Hepatic mitochondrial function loss is associated with cancer cachexia pathology in vivo. Here, we examined if hepatic mitochondrial defects observed in vivo in the cachexic liver also recapitulate during the in vitro treatment of mouse hepatocytes with tumor conditioned media. In vitro experiments were combined with proteome-wide expression analysis of cachexic liver tissue curated for mitochondrial dynamics and quality control proteins, to determine the fidelity of hepatic mitochondrial maladaptation in cancer cachexia pathology. AML12 hepatocytes were exposed to colon-26 (C26) and Lewis lung carcinoma (LLC) conditioned media for 6-72 h and assayed for cell viability, membrane potential, respiratory function, H2O2 production, total ROS/RNS, and mitochondrial dynamics and quality control proteins by immunoblotting. Liver tissue from cachexic C26 mice was analyzed by TMT-based quantitative proteomics for in vivo comparison. Cell viability, membrane potential, H2O2 production, total ROS/RNS, and respiration were decreased 48-72 h after exposure to C26 and/or LLC. Protein expression of treated hepatocytes and cachexic liver tissue showed altered mitochondrial dynamics and quality control, in a manner that suggests limited fusion and content mixing, but also impaired ability to fragment and clear damaged mitochondria. Two strategies to maintain mitochondrial health, therefore, may not be functioning sufficiently in the cachexic liver. Together these findings imply adverse effects of C26 and LLC exposure on hepatocyte health, due to impaired mitochondrial function and remodeling. Exposure of mouse hepatocytes to tumor conditioned media models aspects of cachexic liver mitochondria dysfunction in vivo and validates the importance of hepatic mitochondrial maladaptation in cancer cachexia pathology.

Distinct glycolytic pathway regulation in liver, tumour and skeletal muscle of mice with cancer cachexia.

Energetically inefficient inter-organ substrate shuttles are proposed contributors to cachexia-related weight loss. Here, we examined glycolytic pathway metabolites, enzyme activity and transport proteins in skeletal muscle, liver and tumours of mice with cachexia-related weight loss induced by colon-26 cancer cells. Skeletal muscle of cachexic mice had increased [L-lactate]/[pyruvate], LDH activity and lactate transporter MCT1. Cachexic livers also showed increased MCT1. This is consistent with the proposal that the rate of muscle-derived lactate shuttling to liver for use in gluconeogenesis is increased, that is, an increased Cori cycle flux in weight-losing cachexic mice. A second shuttle between liver and tumour may also contribute to disrupted energy balance and weight loss. We found increased high-affinity glucose transporter GLUT1 in tumours, suggesting active glucose uptake, tumour MCT1 detection and decreased intratumour [L-lactate]/[pyruvate], implying increased lactate efflux and/or intratumour lactate oxidation. Last, high [L-lactate]/[pyruvate] and MCT1 in cachexic muscle provides a potential muscle-derived lactate supply for the tumour (a 'reverse Warburg effect'), supporting tumour growth and consequent cachexia. Our findings suggest several substrate shuttles among liver, skeletal muscle and tumour contribute to metabolic disruption and weight loss. Therapies that aim to normalize dysregulated substrate shuttling among energy-regulating tissues may alleviate unintended weight loss in cancer cachexia. SIGNIFICANCE OF THE STUDY: Cachexia is a serious complication of cancer characterized by severe weight loss, muscle atrophy and frailty. Cachexia occurs in roughly half of all cancer patients, and in up to 80% of patients with advanced disease. Cachexia independently worsens patient prognosis, lowers treatment efficacy, increases hospitalization cost and length of stay, and accounts for 20-30% of cancer-related deaths. There are no effective treatments. Our findings suggest several substrate shuttles among liver, skeletal muscle and tumour contribute to metabolic disruption and weight loss in cancer cachexia. Identifying therapies that normalize dysregulated substrate shuttling among energy-regulating tissues may protect against cachexia-related weight loss.

Hepatic proteome analysis reveals altered mitochondrial metabolism and suppressed acyl-CoA synthetase-1 in colon-26 tumor-induced cachexia.

Cachexia is a life-threatening complication of cancer traditionally characterized by weight loss and muscle dysfunction. Cachexia, however, is a systemic disease that also involves remodeling of nonmuscle organs. The liver exerts major control over systemic metabolism, yet its role in cancer cachexia is not well understood. To advance the understanding of how the liver contributes to cancer cachexia, we used quantitative proteomics and bioinformatics to identify hepatic pathways and cellular processes dysregulated in mice with moderate and severe colon-26 tumor-induced cachexia; ~300 differentially expressed proteins identified during the induction of moderate cachexia were also differentially regulated in the transition to severe cachexia. KEGG pathway enrichment revealed representation by oxidative phosphorylation, indicating altered hepatic mitochondrial function as a common feature across cachexia severity. Glycogen catabolism was also observed in cachexic livers along with decreased pyruvate dehydrogenase protein X component (Pdhx), increased lactate dehydrogenase A chain (Ldha), and increased lactate transporter Mct1. Together this suggests altered lactate metabolism and transport in cachexic livers, which may contribute to energetically inefficient interorgan lactate cycling. Acyl-CoA synthetase-1 (ACSL1), known for activating long-chain fatty acids, was decreased in moderate and severe cachexia based on LC-MS/MS analysis and immunoblotting. ACSL1 showed strong linear relationships with percent body weight change and muscle fiber size (R2 = 0.73-0.76, P < 0.01). Mitochondrial coupling efficiency, which is compromised in cachexic livers to potentially increase energy expenditure and weight loss, also showed a linear relationship with ACSL1. Findings suggest altered mitochondrial and substrate metabolism of the liver in cancer cachexia, and possible hepatic targets for intervention.

Correction to: Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3.

An amendment to this paper has been published and can be accessed via the original article.

Tissue-specific dysregulation of mitochondrial respiratory capacity and coupling control in colon-26 tumor-induced cachexia.

In addition to skeletal muscle dysfunction, cancer cachexia is a systemic disease involving remodeling of nonmuscle organs such as adipose and liver. Impairment of mitochondrial function is associated with multiple chronic diseases. The tissue-specific control of mitochondrial function in cancer cachexia is not well defined. This study determined mitochondrial respiratory capacity and coupling control of skeletal muscle, white adipose tissue (WAT), and liver in colon-26 (C26) tumor-induced cachexia. Tissues were collected from PBS-injected weight-stable mice, C26 weight-stable mice and C26 mice with moderate (10% weight loss) and severe cachexia (20% weight loss). The respiratory control ratio [(RCR) an index of oxidative phosphorylation (OXPHOS) coupling efficiency] was low in WAT during the induction of cachexia because of high nonphosphorylating LEAK respiration. Liver RCR was low in C26 weight-stable and moderately cachexic mice because of reduced OXPHOS. Liver RCR was further reduced with severe cachexia, where Ant2 but not Ucp2 expression was increased. Ant2 was inversely correlated with RCR in the liver (r = -0.547, P < 0.01). Liver cardiolipin increased in moderate and severe cachexia, suggesting this early event may also contribute to mitochondrial uncoupling. Impaired skeletal muscle mitochondrial respiration occurred predominantly in severe cachexia, at complex I. These findings suggest that mitochondrial function is subject to tissue-specific control during cancer cachexia, whereby remodeling in WAT and liver arise early and may contribute to altered energy balance, followed by impaired skeletal muscle respiration. We highlight an under-recognized role of liver and WAT mitochondrial function in cancer cachexia and suggest mitochondrial function of multiple tissues to be therapeutic targets.

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich's ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.

Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3.

BACKGROUND: STAT3 is increasingly becoming known for its non-transcriptional regulation of mitochondrial bioenergetic function upon activation of its S727 residue (S727-STAT3). Lengthy mitochondrial dysfunction can lead to cell death. We tested whether an integrin-FAK-STAT3 signaling pathway we recently discovered regulates mitochondrial function and cell survival, and treatments thereof. METHODS: Cultured mouse brain bEnd5 endothelial cells were treated with integrin, FAK or STAT3 inhibitors, FAK siRNA, as well as integrin and STAT3 activators. STAT3 null cells were transfected with mutant STAT3 plasmids. Outcome measures included oxygen consumption rate for mitochondrial bioenergetics, Western blotting for protein phosphorylation, mitochondrial membrane potential for mitochondrial integrity, ROS production, and cell counts. RESULTS: Vitronectin-dependent mitochondrial basal respiration, ATP production, and maximum reserve and respiratory capacities were suppressed within 4 h by RGD and αvß3 integrin antagonist peptides. Conversely, integrin ligands vitronectin, laminin and fibronectin stimulated mitochondrial function. Pharmacological inhibition of FAK completely abolished mitochondrial function within 4 h while FAK siRNA treatments confirmed the specificity of FAK signaling. WT, but not S727A functionally dead mutant STAT3, rescued bioenergetics in cells made null for STAT3 using CRISPR-Cas9. STAT3 inhibition with stattic in whole cells rapidly reduced mitochondrial function and mitochondrial pS727-STAT3. Stattic treatment of isolated mitochondria did not reduce pS727 whereas more was detected upon phosphatase inhibition. This suggests that S727-STAT3 is activated in the cytoplasm and is short-lived upon translocation to the mitochondria. FAK inhibition reduced pS727-STAT3 within mitochondria and reduced mitochondrial function in a non-transcriptional manner, as shown by co-treatment with actinomycin. Treatment with the small molecule bryostatin-1 or hepatocyte growth factor (HGF), which indirectly activate S727-STAT3, preserved mitochondrial function during FAK inhibition, but failed in the presence of the STAT3 inhibitor. FAK inhibition induced loss of mitochondrial membrane potential, which was counteracted by bryostatin, and increased superoxide and hydrogen peroxide production. Bryostatin and HGF reduced the substantial cell death caused by FAK inhibition over a 24 h period. CONCLUSION: These data suggest that extracellular matrix molecules promote STAT3-dependent mitochondrial function and cell survival through integrin-FAK signaling. We furthermore show a new treatment strategy for cell survival using S727-STAT3 activators.

Altered Cerebellar Circuitry following Thoracic Spinal Cord Injury in Adult Rats.

Cerebellar function is critical for coordinating movement and motor learning. However, events occurring in the cerebellum following spinal cord injury (SCI) have not been investigated in detail. We provide evidence of SCI-induced cerebellar synaptic changes involving a loss of granule cell parallel fiber input to distal regions of the Purkinje cell dendritic tree. This is accompanied by an apparent increase in synaptic contacts to Purkinje cell proximal dendrites, presumably from climbing fibers originating in the inferior olive. We also observed an early stage injury-induced decrease in the levels of cerebellin-1, a synaptic organizing molecule that is critical for establishing and maintaining parallel fiber-Purkinje cell synaptic integrity. Interestingly, this transsynaptic reorganizational pattern is consistent with that reported during development and in certain transgenic mouse models. To our knowledge, such a reorganizational event has not been described in response to SCI in adult rats. Regardless, the novel results of this study are important for understanding SCI-induced synaptic changes in the cerebellum, which may prove critical for strategies focusing on promoting functional recovery.

Differential proteomic analysis of acute contusive spinal cord injury in rats using iTRAQ reagent labeling and LC-MS/MS.

In this experimental study, differential labeling with isobaric tags for relative and absolute quantitation (iTRAQ) reagents followed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS) proteomic approach was used to investigate differences in the proteome of rat spinal cord at 24 h following a moderate contusion injury. Spinal cord protein samples from the injury epicenter (or from sham controls) were trypsinized and differentially labeled with iTRAQ isotopic reagents. The differentially labeled samples were then combined into one sample mixture, separated by LC, and analyzed using MS/MS. Proteins were quantified by comparing the peak areas of iTRAQ reporter fragment ions in MS/MS spectra. The outcome of this analysis revealed that proteins involved in ubiquitination, endocytosis and exocytosis, energy metabolism, inflammatory response, oxidative stress, cytoskeletal disruption, and vascular damage were significantly altered at 24 h following spinal cord injury (SCI). This study demonstrates the utility of the iTRAQ method in proteomic studies and provides further insights into secondary events that occur during acute times following SCI.

Mitochondrial stress and mitokines in aging.

Mitokines are signaling molecules that enable communication of local mitochondrial stress to other mitochondria in distant cells and tissues. Among those molecules are FGF21, GDF15 (both expressed in the nucleus) and several mitochondrial-derived peptides, including humanin. Their responsiveness to mitochondrial stress induces mitokine-signaling in response for example to exercise, following mitochondrial challenges in skeletal muscle. Such signaling is emerging as an important mediator of exercise-derived and dietary strategy-related molecular and systemic health benefits, including healthy aging. A compensatory increase in mitokine synthesis and secretion could preserve mitochondrial function and overall cellular vitality. Conversely, resistance against mitokine actions may also develop. Alterations of mitokine-levels, and therefore of mitokine-related inter-tissue cross talk, are associated with general aging processes and could influence the development of age-related chronic metabolic, cardiovascular and neurological diseases; whether these changes contribute to aging or represent "rescue factors" remains to be conclusively shown. The aim of the present review is to summarize the expanding knowledge on mitokines, the potential to modulate them by lifestyle and their involvement in aging and age-related diseases. We highlight the importance of well-balanced mitokine-levels, the preventive and therapeutic properties of maintaining mitokine homeostasis and sensitivity of mitokine signaling but also the risks arising from the dysregulation of mitokines. While reduced mitokine levels may impair inter-organ crosstalk, also excessive mitokine concentrations can have deleterious consequences and are associated with conditions such as cancer and heart failure. Preservation of healthy mitokine signaling levels can be achieved by regular exercise and is associated with an increased lifespan.

Comprehensive evaluation of mitochondrial redox profile, calcium dynamics, membrane integrity and apoptosis markers in a preclinical model of severe penetrating traumatic brain injury.

Traumatic Brain Injury (TBI) is caused by the external physical assaults damages the brain. It is a heterogeneous disorder that remains a leading cause of death and disability in the military and civilian population of the United States. Preclinical investigations of mitochondrial responses in TBI have ascertained that mitochondrial dysfunction is an acute indicator of cellular damage and plays a pivotal role in long-term injury progression through cellular excitotoxicity. The current study was designed to provide an in-depth evaluation of mitochondrial endpoints with respect to redox and calcium homeostasis, and cell death responses following penetrating TBI (PTBI). To evaluate these pathological cascades, anesthetized adult male rats (N = 6/group) were subjected to either 10% unilateral PTBI or Sham craniectomy. Animals were euthanized at 24 h post-PTBI, and purified mitochondrial fractions were isolated from the brain injury core and perilesional areas. Overall, increased reactive oxygen and nitrogen species (ROS/RNS) production, and elevated oxidative stress markers such as 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT), and protein carbonyls (PC) were observed in the PTBI group compared to Sham. Mitochondrial antioxidants such as glutathione, peroxiredoxin (PRX-3), thioredoxin (TRX), nicotinamide adenine dinucleotide phosphate (NADPH), superoxide dismutase (SOD), and catalase (CAT) levels were significantly decreased after PTBI. Likewise, PTBI mitochondria displayed significant loss of Ca2+ homeostasis, early opening of mitochondrial permeability transition pore (mPTP), and increased mitochondrial swelling. Both, outer and inner mitochondrial membrane integrity markers, such as voltage-dependent anion channels (VDAC) and cytochrome c (Cyt C) expression were significantly decreased following PTBI. The apoptotic cell death was evidenced by significantly decreased B-cell lymphoma-2 (Bcl-2) and increased glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression after PTBI. Collectively, current results highlight the comprehensive picture of mitochondria-centric acute pathophysiological responses following PTBI, which may be utilized as novel prognostic indicators of disease progression and theragnostic indicators for evaluating neuroprotection therapeutics following TBI.

An exploratory investigation of apoptotic and autophagic responses in peripheral blood mononuclear cells following maximal aerobic exercise in obese individuals.

Autophagy is a critical molecular process in promoting cell survival against apoptosis. This study examined whether maximal aerobic exercise-mediated apoptosis in obesity might be underlying the involvement of autophagy in the peripheral blood mononuclear cells (PBMCs). Twelve healthy male subjects (6 obese and 6 normal-weight) were recruited to participate in a maximal graded exercise test on a treadmill. Obese subjects exhibited a significantly lower Bax, but a higher Bcl-2 protein level in conjunction with a reduced Bax/Bcl-2 AUCi compared to normal-weight subjects following exercise. Furthermore, a greater LC3-II/LC3-I ratio and LC3-II/LC3-I AUCi was observed in obese subjects compared to normal-weight subjects. LC3-II/LC3-I AUCi was also positively associated with obesity-associated parameters (BMI, waist/hip circumference, and fasting insulin level), but was negatively correlated with Bax/Bcl-2 AUCi. These findings demonstrate that maximal aerobic exercise differentially mediates the intrinsic apoptotic pathway and autophagic activity in human PBMCs isolated from obese compared to normal-weight individuals.

Resistance Training Modulates Reticulum Endoplasmic Stress, Independent of Oxidative and Inflammatory Responses, in Elderly People.

Aging is related to changes in the redox status, low-grade inflammation, and decreased endoplasmic reticulum unfolded protein response (UPR). Exercise has been shown to regulate the inflammatory response, balance redox homeostasis, and ameliorate the UPR. This work aimed to investigate the effects of resistance training on changes in the UPR, oxidative status, and inflammatory responses in peripheral blood mononuclear cells of elderly subjects. Thirty elderly subjects volunteered to participate in an 8-week resistance training program, and 11 youth subjects were included for basal assessments. Klotho, heat shock protein 60 (HSP60), oxidative marker expression (catalase, glutathione, lipid peroxidation, nuclear factor erythroid 2-related factor 2, protein carbonyls, reactive oxygen species, and superoxide dismutase 1 and 2), the IRE1 arm of UPR, and TLR4/TRAF6/pIRAK1 pathway activation were evaluated before and following training. No changes in the HSP60 and Klotho protein content, oxidative status markers, and TLR4/TRAF6/pIRAK1 pathway activation were found with exercise. However, an attenuation of the reduced pIRE1/IRE1 ratio was observed following training. Systems biology analysis showed that a low number of proteins (RPS27A, SYVN1, HSPA5, and XBP1) are associated with IRE1, where XBP1 and RPS27A are essential nodes according to the centrality analysis. Additionally, a gene ontology analysis confirms that endoplasmic reticulum stress is a key mechanism modulated by IRE1. These findings might partially support the modulatory effect of resistance training on the endoplasmic reticulum in the elderly.

High glucose upregulates upstream stimulatory factor 2 in human renal proximal tubular cells through angiotensin II-dependent activation of CREB.

BACKGROUND/AIMS: We have previously demonstrated that a transcription factor, upstream stimulatory factor 2 (USF2), regulates glucose-induced thrombospondin 1 expression and transforming growth factor-ß activity in mesangial cells, and plays an important role in diabetic glomerulopathy. In this study, we determined whether USF2 expression in renal proximal tubular cells is regulated by glucose and contributes to diabetic tubulointerstitial fibrosis. METHODS: Human renal proximal tubular cells (HK-2 cells) were treated with normal- or high-glucose medium for 24 h. After treatment, real-time PCR or immunoblotting was used to determine the expression of USF2 and other components of the renin-angiotensin system in HK-2 cells. RESULTS: High glucose upregulated USF2 expression and increased extracellular matrix accumulation in HK-2 cells; both were inhibited by siRNA-mediated USF2 knockdown. In addition, high glucose stimulated angiotensinogen and renin expression, increased renin activity, and resulted in increased angiotensin II formation. Treatment of HK-2 cells with an angiotensin II receptor 1 (AT1) blocker--losartan--prevented high-glucose-induced USF2 expression and high-glucose-enhanced phosphorylation of CREB (cAMP response element-binding protein). CONCLUSION: Our data established that high glucose stimulated USF2 expression in HK-2 cells, at least in part, through angiotensin II-AT1-dependent activation of CREB, which can contribute to diabetic tubulointerstitial fibrosis.

Resistance Training Diminishes the Expression of Exosome CD63 Protein without Modification of Plasma miR-146a-5p and cfDNA in the Elderly.

Aging-associated inflammation is characterized by senescent cell-mediated secretion of high levels of inflammatory mediators, such as microRNA (miR)-146a. Moreover, a rise of circulating cell-free DNA (cfDNA) is also related to systemic inflammation and frailty in the elderly. Exosome-mediated cell-to-cell communication is fundamental in cellular senescence and aging. The plasma changes in exercise-promoted miR-146a-5p, cfDNA, and exosome release could be the key to facilitate intercellular communication and systemic adaptations to exercise in aging. Thirty-eight elderly subjects (28 trained and 10 controls) volunteered in an 8-week resistance training protocol. The levels of plasma miR-146a-5p, cfDNA, and exosome markers (CD9, CD14, CD63, CD81, Flotillin [Flot]-1, and VDAC1) were measured prior to and following training. Results showed no changes in plasma miR-146a-5p and cfDNA levels with training. The levels of exosome markers (Flot-1, CD9, and CD81) as well as exosome-carried proteins (CD14 and VDAC1) remained unchanged, whereas an attenuated CD63 response was found in the trained group compared to the controls. These findings might partially support the anti-inflammatory effect of resistance training in the elderly as evidenced by the diminishment of exosome CD63 protein expression, without modification of plasma miR-146a-5p and cfDNA.

Purified c-phycoerythrin: safety studies in rats and protective role against permanganate-mediated fibroblast-DNA damage.

We have evaluated in vitro cytotoxicity of cyanobacterial phycoerythrin (C-PE) on three human cell lines by cell proliferation and neutral red uptake assays. No toxic effects of C-PE were observed to any of the cell lines tested. The protective role of purified C-PE to potassium permanganate-mediated human fibroblast-DNA damage was assessed by comet assay at 0 (control), 10 and 20 microg C-PE ml(-1) doses in pre-, simultaneous and post-mutagen exposure conditions. Significant DNA damage was detected only in post-mutagen exposure conditions. Our findings confirmed that the C-PE is non-toxic and provides protection against permanganate-mediated DNA damage. The preliminary acute (2000 mg C-PE kg(-1) body weight, b.w.) and 90 day sub-chronic (0, 5, 15 and 25 mg C-PE kg(-1) b.w./day) oral toxicity studies of purified C-PE in male albino rats showed no mortality or treatment-related major clinical signs, and all the doses of C-PE were well tolerated. The no observed adverse effect level and no observed effect level were found to be 15 and 5 mg C-PE kg(-1) b.w./day respectively.

Chlorophytum borivilianum as potential terminator of free radicals in various in vitro oxidation systems.

Chlorophytum borivilianum is a very popular herb in traditional Indian medicine and used as a potent "Rasayana" drug in "Ayurveda" as a rejuvenator. Currently, a large body of evidence supports the key role of free radicals in diverse pathological conditions such as aging and atherosclerosis. The present investigation essentially focuses on the comprehensive account of in vitro antioxidant activity exerted by C.borivilianum root extracts (i.e., aqueous and ethanolic), to clarify the pharmacological antagonism of chemicals/metals-mediated oxidation. Graded-dose (25 to 1000 microg/ml) of aqueous extract exhibited higher antioxidant potency as evidenced by powerful nitric oxide, superoxide, hydroxyl, DPPH and ABTS(*+) radicals scavenging activity along with reducing capacity (Fe(3+)/ferricyanide complex and FRAP assays), metal chelating ability, as well as markedly suppressed the lipid peroxidation in mitochondrial fractions as compared to ethanolic extract. Further, aqueous extract significantly decreased (P < 0.05) copper-mediated human serum and kinetics of LDL oxidation, as demonstrated by prolongation of lag phase time with decline of oxidation rate, conjugated dienes, lipid hydroperoxides and thiobarbituric acid reactive substances. In addition, the total polyphenol and flavonoid contents of aqueous extract were higher than that of ethanolic extract, which indicated a positive correlation between antioxidant activity and contents of total phenols. The IC(50) values of both extracts were also compared with appropriate antioxidant standards. Overall, aqueous extract of C.borivilianum root has significant powerful antioxidant activity and may favorably affect atherosclerosis risk status by reducing LDL oxidation susceptibility.

Aerobic Training Down-Regulates Pentraxin 3 and Pentraxin 3/Toll-Like Receptor 4 Ratio, Irrespective of Oxidative Stress Response, in Elderly Subjects.

Reactive oxygen and nitrogen species-mediated cellular aging has been linked to diseases such as atherothrombosis and cancer. Although pentraxin 3 (PTX3) is associated with aging-related diseases via TLR4-dependent anti-inflammatory effects, its relationship with oxidative stress in aging remains to be elucidated. Exercise is proposed as the key intervention for health maintenance in the elderly. This study aimed to examine the association of PTX3 levels with changes in oxidative stress in both plasma and peripheral blood mononuclear cells (PBMCs), following aerobic training in elderly adults. Nine trained and five controls participated in an eight-week aerobic training protocol. Enzyme-linked immunosorbent assay (ELISA) and Western blot analyses were used to determine PTX3, toll-like receptor 4 (TLR4), and levels of oxidative stress biomarkers [3-nitrotyrosine (3NT), 4-hydroxynonenal (4-HNE), reduced glutathione (GSH), protein carbonyl (PC), reactive oxygen/ nitrogen species (ROS/RNS), trolox equivalent antioxidant capacity (TEAC)] in plasma and/or PBMCs. Results showed a down-regulation of PTX3 expression in PBMCs following aerobic training, along with decreased PTX3/TLR4 ratios. Oxidative stress responses in PBMCs remained unchanged with the exercise protocol. Comparable levels of plasma PTX3 and oxidative stress biomarkers were observed in trained vs. control groups. No correlation was found between PTX3 and any oxidative stress biomarkers following training. These findings demonstrated the down-regulation of PTX3 and PTX3/TLR4 ratio, irrespective of oxidative stress response, in elderly adults following eight weeks of aerobic training.